Composition for promoting cartilage formation or repair comprising a nell gene product and method of treating cartilage-related conditions using such composition

ABSTRACT

Provided herein is a composition for cartilage formation or regeneration comprising a NELL gene product and a method of treating cartilage-related conditions using such a composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/527,786, filed on Sep. 28, 2005, which is a U.S. National Phase ofinternational application No. PCT/US 2003/29281, filed on Sep. 15, 2003,which claims priority to U.S. provisional application No. 60/410,846,filed on Sep. 13, 2002; U.S. Ser. No. 10/527,786 is now abandoned.

This application is also a continuation-in-part of U.S. application Ser.No. 10/544,553, filed on May 15, 2006, now U.S. Pat. No.7,544,486, whichis a U.S. National Phase of PCT application PCT/US2004/003808, filed onFeb. 9, 2004, which claims priority to U.S. provisional application No.60/445,672, filed on Feb. 7, 2003, and PCT/US2003/29281, filed on Sep.15, 2003, the teachings of which are incorporated herein by reference.This application is also a continuation-in-part of internationalapplication No. PCT/US2006/005473, filed on Feb. 16, 2006, which claimspriority to U.S. Provisional Application No. 60/653,722 filed on Feb.16, 2005. This application is also a continuation-in-part of U.S.application Ser. No. 11/392,294, filed on Mar. 28, 2006, which is acontinuation application of U.S. application Ser. No. 09/412,297, filedon Oct. 5, 1999, issued as U.S. Pat. No. 7,052,856.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Part of this work was supported by NIH/NIDR grant number DE9400 andCRC/NIH grant number RR00865, NIH/NIDCR RO3 DE 014649-01, NIH/NIDCRK23DE00422, NIH DE016107-01, and NIH/SBIR R43-DE016781-01. TheGovernment of the United States of America has certain rights in thisinvention.

FIELD OF THE INVENTION

The invention generally relates to a composition for cartilage formationor regeneration.

BACKGROUND OF THE INVENTION

Growth factors are substances, such as peptides, which affect the growthand differentiation of defined populations of cells in vivo or in vitro.

Cartilage is a type of dense connective tissue. It is composed ofchondrocytes which are dispersed in a firm gel-like matrix. Cartilage isavascular (contains no blood vessels) and nutrients are diffused throughthe matrix. Cartilage is found in the joints, the rib cage, the ear, thenose, in the throat and between intervertebral disks. There are threemain types of cartilage: hyaline (e.g., costal cartilages, thecartilages of the nose, trachea, and bronchi, and the articularcartilages of joints), elastic (e.g., external ear, external auditorymeatus, part of the Eustachian tube, epiglottis, and in some of thelaryngeal cartilages) and fibrocartilage [e.g. meniscus (e.g., wristtriangular fibrocartilage complex, knee meniscus), intervertebral discs,temporomandibular joint disc, the pubic symphysis, and in some tendonsand ligaments at their attachment to bones. One of the main purposes ofcartilage is to provide a framework upon which bone deposition couldbegin (i.e., during endochondral ossification). Another importantpurpose of cartilage is to provide smooth surfaces for the movement ofarticulating bones. For example, articular cartilage, most notably thatwhich is found in the knee joint, is generally characterized by very lowfriction, high wear resistance, and poor regenerative qualities. It isresponsible for much of the compressive resistance and load bearingqualities of the knee joint and, without it, walking is painful toimpossible. Yet another important purpose of cartilage is to provide,firm, yet flexible support (e.g., nasal cartilage, spinal discs,tracheal cartilage, knee meniscus, bronchial cartilage). For instance,cartilage such as the meniscus plays a crucial role in joint stability,lubrication, and force transmission. Under a weight bearing load, themeniscus maintains the balanced position of the femur on the tibia anddistributes the compressive forces by increasing the surface contactarea, thereby decreasing the average stress two to three times.Additionally, the menisci interact with the joint fluid to produce acoefficient of friction that is five times as slick as ice on ice. Inanother example, the intervertebral disc has several importantfunctions, including functioning as a spacer, as a shock absorber, andas a motion unit. The gelatinous central portion of the disc is calledthe nucleus pulposus. It is composed of 80-90% water. The solid portionof the nucleus is Type II collagen and non-aggregated proteoglycans. Theouter ligamentous ring around the nucleus pulposus is called the annulusfibrosus, which hydraulically seals the nucleus, and allows intradiscalpressures to rise as the disc is loaded. The annulus has overlappingradial bands, not unlike the plies of a radial tire, and this allowstorsional stresses to be distributed through the annulus under normalloading without rupture. The disc functions as a hydraulic cylinder. Theannulus interacts with the nucleus. As the nucleus is pressurized, theannular fibers serve a containment function to prevent the nucleus frombulging or herniating.

Cartilage can be damaged by wear, injury or diseases. As we age, thewater and protein content of the body's cartilage changes. This changeresults in weaker, more fragile and thin cartilage. Osteoarthritis is acommon condition of cartilage failure that can lead to limited range ofmotion, bone damage and invariably, pain. Due to a combination of acutestress and chronic fatigue, osteoarthritis directly manifests itself ina wearing away of the articulating surface and, in extreme cases, bonecan be exposed in the joint. In another example, loss of the protectivestabilizing meniscus leads to increased joint laxity or abnormal motionsthat lead to joint instability. The excessive motion and narrowedcontact area promotes early arthritic changes. At the cellular level,there is initially a loss of cells from the superficial layer of thearticular cartilage followed by cartilage splitting, subsequent thinningand erosion occurs, and finally protrusion of the underlying raw bone.The earliest arthritic changes have been noted three weeks after loss ofthe entire meniscus. In yet another example, because both the discs andthe joints that stack the vertebrae (facet joints) are partly composedof cartilage, these areas are subject to wear and tear over time(degenerative changes). As the inner nucleus dehydrates, the disc spacenarrows, and redundant annular ligaments bulge. With progressive nucleardehydration, the annular fibers can crack and tear. Loss of normal softtissue tension may allow the spinal segment to sublux (e.g. partialdislocation of the joint), leading to osteophyte formation (bone spurs),foraminal narrowing, mechanical instability, and pain. If the annularfibers stretch or rupture, allowing the pressurized nuclear material tobulge or herniate and compress neural tissues, pain and weakness mayresult. This is the condition called a pinched nerve, slipped disc, orherniated disc. Radiculopathy refers to nerve irritation caused bydamage to the disc between the vertebrae. Mechanical dysfunction mayalso cause disc degeneration and pain (e.g. degenerative disc disease).For example, the disc may be damaged as the result of some trauma thatoverloads the capacity of the disc to withstand increased forces passingthrough it, and inner or outer portions of the annular fibers may tear.These tom fibers may be the focus for inflammatory response when theyare subjected to increased stress, and may cause pain directly, orthrough the compensatory protective spasm of the deep paraspinalmuscles.

There are several different repair options available for cartilagedamage or failure. Osteoarthritis is the second leading cause ofdisability in the elderly population in the United States. It is adegenerative disorder that generally starts off relatively mild andescalates with time and wear. For those patients experiencing mild tomoderate symptoms, the disorder can be dealt with by severalnon-surgical treatments. The use of braces and drug therapies, such asanti-inflammatories (ex. diclofenac, ibuprofen, and naproxen), COX-2selective inhibitors, hydrocortisone, glucosamine, and chondroitinsulfate, have been shown to alleviate the pain caused by cartilagedeficiency and some claim they can slow the degenerative process.

Most surgical treatments for articular cartilage, short of total jointreplacement, can be divided into various treatment groups. Treatmentsthat remove the diseased and undermined cartilage with an aim to stopinflammation and pain include shaving (chondrectomy) and debridement.Another group of treatments consists of a range of abrasive proceduresaimed at triggering cartilage production, such as drilling,microfracture surgery, chondroplasty, and spongialization. Abrasion,drilling, and microfracture originated 20 years ago. They rely on thephenomenon of spontaneous repair of the cartilage tissue followingvascular injury to the subchondral plate of the bone. Laser assistedtreatments, currently experimental, compose another category; theycombine the removal of diseased cartilage with cartilage reshaping andalso induce cartilage proliferation. Additional treatments includeautologous cartilage implants (e.g., Carticel by Genzyrne). Othertreatments, more applicable to meniscal cartilage, include earlysurgical intervention and suture repair of tom structures or allograftmeniscus transplantation in severe injury cases.

Although the overwhelming majority of patients with a herniated disc andsciatica heal without surgery, if surgery is indicated proceduresinclude removal of the herniated disc with laminotomy (producing a smallhole in the bone of the spine surrounding the spinal cord), laminectomy(removal of the bony wall adjacent to the nerve tissues), by needletechnique through the skin (percutaneous discectomy), disc-dissolvingprocedures (chemonucleolysis), and others. For patients with mechanicalpain syndrome, unresponsive to conservative treatment, and disabling tothe individual's way of life, the problem can be addressed by spinalfusion, intradiscal electrothermal coagulation (or annuloplasty),posterior dynamic stabilization, artificial disc technologies, or stillexperimental disc regeneration therapies using various molecular basedtherapies delivered using proteins, peptides, gene therapies, ornucleotides. Although numerous methods have been described for treatmentof cartilage problems, it is clear that many are artificial ormechanically based solutions that do not seek to recreate normalcartilage tissue biology. Therefore, there is a need for methods forstimulating cartilage formation.

The embodiments described below address the above identified issues andneeds.

SUMMARY OF THE INVENTION

The present invention is related to agents and methods for inducingcartilage formation or repair using a NELL peptide or related agent(collectively referred as “agent”). The composition can include a NELLpeptide, a Nell-like molecule, and optionally at least one other activeagent, cells, and biocompatible material implanted for the purpose ofcartilage repair (i.e., hyaline cartilage, elastic cartilage, orfibrocartilage).

In some embodiments, the present invention provides a composition thatcontains an effective amount of at least one agent for either directlyor indirectly promoting the generation of cartilage for treating,preventing or ameliorating a cartilage related medical condition. One ofthe agents for direct promotion of cartilage generation can be NELLpeptides or NELL-based gene therapy or NELL-gene product enhancersapplied to chondrogenic cells such as, but not limited to,chondroblasts, chondrocytes, or chondroprogenitor cells, adult andembryonic stem cells, bone marrow cells, bone marrow stromal cells,mesenchymal cells, a fibroblast, or adipose derived cells. The agent forindirect promotion of cartilage generation (e.g., through inducingchondroblast/chondrocyte differentiation) can be, e.g., one of NELLpeptide, or agonists of NELL peptide receptors.

In some embodiments, the composition can include, e.g., one or moreinhibitors or antagonists of NELL peptide receptors, high dose NELLpeptides, or combinations thereof. Such a composition is effective forinhibition of chondrogenic differentiation by inhibiting potential orcommitted chondrogenic cells such as, but not limited to, osteoblasts,osteoprogenitor cells, stem cells, bone marrow cells, fibroblasticcells, dural cells, periosteal cells, pericytes, and/or muscle cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows increased cartilage maturation and hypertrophy in femoralhead cartilage of NELL1 overexpression mice compared with wild typelittermate. On the left is wild type newborn femoral head cartilagedemonstrating small, less mature chondrocytes in the femoral head. Onthe right is the NELL1 over-expression transgenic mice demonstratingwell differentiated, more mature, hypertrophic chondrocytes presentthroughout the femoral head with large nuclei and vacuoles present.

FIGS. 2A-2F show increased meniscus development in E18 NELL1overexpression mice compared with wild type littermate. FIGS. 2A and 2Bwith arrows pointing at the meniscus between the femoral and tibialcartilage head in wild type (FIG. 2A) and NELL1 overexpression (FIG. 2B)animals. FIGS. 2C and 2D are higher magnification views of FIGS. 2A and2B. FIG. 2E is a higher magnification of the wild type control shown in2C demonstrating less differentiated chondrocytes with minimalhypertrophy. FIG. 2F is a higher magnification of the NELL1overexpression animal shown in 2D demonstrating significantly moredifferentiated chondrocyte in the cartilage matrix. Vacuoles in thehypertrophic chondrocytes are observed indicating well differentiationof chondrocyte in the meniscus.

FIGS. 3A and 3B show adenovirus transduction of goat primarychondrocytes isolated from auricular cartilage. FIG. 3A shows theefficiency of adenoviral (Ad) transduction with significant number ofpositively stained cells expressing beta-galactosidase. FIG. 3B is aWestern gel demonstrating significant NELL1 protein expression in theAdNELL1 transduced goat chondrocytes (relative to beta-actin controls)and no NELL1 protein expression in Ad BMP2 or AdLacZ (control)transduced goat chondrocytes.

FIGS. 4A and 4B show gross appearance of AdNELL1, AdBMP2, or AdLacZ(control) transduced goat primary chondrocytes 4 weeks afterimplantation/injection into nude mice. NELL1 transduced samples weresignificantly larger than control by both inspection (FIG. 4A) andweight (FIG. 4B). In addition, NELL1 transduced samples did notdemonstrate the discoloration present in the BMP2 transduced samples.

FIGS. 5A-C show micro computed tomography (CT) examination of thesamples shown in FIG. 4. FIG. 5A demonstrates undesirable mineralization(red coloring) in the AdBMP2 transduced specimens but not AdNELL1 orAdLacZ specimens. FIG. 5B demonstrates that NELL1 induces significantlymore cartilage mass than AdLacZ controls. FIG. 5C demonstrates thatAdBMP2 significantly increased density (another indicator ofmineralization) in the specimens.

FIG. 6 shows histologic appearance of AdNELL1, AdBMP2, or AdLacZ(control) transduced goat primary chondrocytes 2 weeks afterimplantation/injection into nude mice. Hematoxylin and eosin (H&E)staining (1^(st) row) shows evidence of increased cartilage formation inthe AdNELL1 and AdBMP2 transduced specimens relative to AdLacZ controls.Alcian blue staining which stains cartilage (2^(nd) row) alsodemonstrates increased cartilage formation in the AdNELL1 and AdBMP2transduced specimens relative to AdLacZ controls. Type X collagen (ColX)immunostaining which stains more mature cartilage cells (3^(rd) row)demonstrates increased staining in the AdNELL1 and AdBMP2 transducedspecimens.

FIG. 7 shows histologic appearance of AdNELL1, AdBMP2, or AdLacZ(control) transduced goat primary chondrocytes 4 weeks afterimplantation/injection into nude mice. H&E staining (1^(st) row) showssignificant cartilage formation in the AdNELL transduced samples with noevidence of bone formation, while AdBMP2 samples show significant boneformation. A small amount of cartilage formation is seen the AdLacZcontrols. Alcian blue staining (2^(nd) row) also demonstratessignificant cartilage formation in the AdNELL transduced samples with noevidence of bone formation, while AdBMP2 samples show significant boneformation and minimal cartilage formation. A small amount of immaturecartilage formation is seen the AdLacZ controls.

FIG. 8 shows immunostaining for bone marker Cbfa1/Runx2 and cartilagemarkers ColX and tenascin in AdNELL1, AdBMP2, or AdLacZ (control)transduced goat primary chondrocytes 4 weeks afterimplantation/injection into nude mice. Tenascin is intimately associatedwith the development of articular cartilage and other permanentcartilages whereas absence or reduced amounts of this matrix proteincharacterize transient cartilages which undergo maturation and arereplaced by bone (Pacifici, M., M. Iwamoto, et al. Tenascin isassociated with articular cartilage development. Dev Dyn 198(2): 123-34,1993). Cbfa1/Runx2 is minimally expressed in cartilaginous AdNELL1 orcontrol AdLacZ transduced samples and moderately expressed in bonyAdBMP2 transduced samples (1^(st) row). ColX is highly expressed andlocalized largely to cells in cartilaginous AdNELL1 samples withoutevidence of bone formation, while ColX is largely associated with theextracelluar matrix rather than cells in the AdBMP2 treated samples(2^(nd) row). Tenascin is highly expressed in AdNELL1 samples andminimally present in AdBMP2 and control AdLacZ samples (3^(rd) row).

FIG. 9 shows immunostaining for endochondral ossification associatedangiogenic growth factor, vascular endothelial growth factor (VEGF), andbone marker osteocalcin (OCN) in AdNELL1, AdBMP2, or AdLacZ (control)transduced goat primary chondrocytes 4 weeks afterimplantation/injection into nude mice. Both VEGF and OCN are notexpressed in cartilaginous AdNELL1 or control AdLacZ transduced samplesand moderately expressed in bony AdBMP2 transduced samples.

FIG. 10 shows the histology of long bone cartilage in NELL-1 overexpression mice. NELL1 is expressed throughout the tibia duringendochondral bone formation including both articular cartilage region(Upper panel) and also the long bone formation region (lower panel).Upper panel demonstrates that NELL1 can modulate and increase cartilagedifferentiation in the articular cartilage region. Accordingly, thesedata show that increased NELL peptide activity directly (e.g., throughaddition of NELL peptides or increased NELL peptide expression) orindirectly (e.g., through addition of NELL peptide enhancers and/or NELLpeptide receptor agonists and/or activators) promotes cartilageformation. In the lower panel, in the long bone shaft region where boneformation originated, increased NELL1 causes cartilage formation andthen hypertrophy and increased bone formation through endochondralossification.

DETAILED DESCRIPTION

The present invention is related to agents and methods for inducingcartilage formation or repair using a NELL peptide or related agent(collectively referred as “agent”). The composition can include a NELLpeptide, a Nell-like molecule, and optionally at least one other activeagent, cells, and biocompatible material implanted for the purpose ofarticular cartilage repair.

In some embodiments, the present invention provides a composition thatcontains an effective amount of at least one agent for either directlyor indirectly promoting the generation of cartilage for treating,preventing or ameliorating a cartilage related medical condition. One ofthe agents for direct promotion of cartilage generation can be NELLpeptides applied to chondrogenic cells such as, but not limited to,chondroblasts, chondrocytes, or chondroprogenitor cells, stem cells,bone marrow cells, a bone marrow stromal cells, a fibroblast, or adiposederived cells. The agent for indirect promotion of cartilage generation(e.g., through inducing chondroblast/chondrocyte differentiation) canbe, e.g., one of NELL peptide, or agonists of NELL peptide receptors.

In some embodiments, the present invention includes a systemic or localapplication of the composition described herein to a mammalian subject(e.g., a human being) to promote cartilage formation or regeneration.

In some embodiments, the composition can include, e.g., one or moreinhibitors or antagonists of NELL peptide receptors, high dose NELLpeptides, or combinations thereof. Such a composition is effective forinhibition of chondrogenic differentiation by inhibiting potential orcommitted chondrogenic cells such as, but not limited to, osteoblasts,osteoprogenitor cells, stem cells, bone marrow cells, fibroblasticcells, dural cells, periosteal cells, pericytes, and/or muscle cells.

The effectiveness of the present invention for cartilage formation orregeneration are shown in FIGS. 1-10.

FIG. 1 shows increased cartilage maturation and hypertrophy in femoralhead cartilage of NELL1 overexpression mice compared with wild typelittermate. On the left is wild type newborn femoral head cartilagedemonstrating small, less mature chondrocytes in the femoral head. Onthe right is the NELL1 over-expression transgenic mice demonstratingwell differentiated, more mature, hypertrophic chondrocytes presentthroughout the femoral head with large nuclei and vacuoles present. Notethe absence of mineralization in the hypertrophied cartilage. Thesestudies demonstrate that NELL1 increases chondrocyte maturation,hypertrophy without necessarily inducing mineralization.

FIGS. 2A-2F show increased meniscus development in E18 NELL1overexpression mice compared with wild type littermate. FIGS. 2A and 2Bwith arrows pointing at the meniscus between the femoral and tibialcartilage head in wild type (FIG. 2A) and NELL1 overexpression (FIG. 2B)animals. FIGS. 2C and 2D are higher magnification views of FIGS. 2A and2B. FIG. 2E is a higher magnification of the wild type control shown in2C demonstrating less differentiated chondrocytes with minimalhypertrophy. FIG. 2F is a higher magnification of the NELL1overexpression animal shown in 2D demonstrating significantly moredifferentiated chondrocyte in the cartilage matrix. Vacuoles in thehypertrophic chondrocytes are observed indicating well differentiationof chondrocyte in the meniscus. This data indicates that Nell-1 canpromote meniscus formation and differentiation.

FIGS. 3A and 3B show adenovirus transduction of goat primarychondrocytes isolated from auricular cartilage. FIG. 3A shows theefficiency of adenoviral (Ad) transduction with significant number ofpositively stained cells expressing beta-galactosidase. FIG. 3B is aWestern gel demonstrating significant NELL1 protein expression in theAdNELL1 transduced goat chondrocytes (relative to beta-actin controls)and no NELL1 protein expression in AdBMP2 or AdLacZ (control) transducedgoat chondrocytes. These studies demonstrate that there is efficientadenoviral transduction and that AdNELL1, but not AdBMP2, increasesNELL1 protein expression.

FIGS. 4A and 4B show gross appearance of AdNELL1, AdBMP2, or AdLacZ(control) transduced goat primary chondrocytes 4 weeks afterimplantation/injection into nude mice. NELL1 transduced samples weresignificantly larger than control by both inspection (FIG. 4A) andweight (FIG. 4B). In addition, NELL1 transduced samples did notdemonstrate the discoloration present in the BMP2 transduced samples.These studies unexpectedly demonstrate that although BMP2 induces alarger tissue mass, the appearance of the induced mass is not consistentwith a purely cartilaginous phenotype.

FIGS. 5A-C show micro computed tomography (CT) examination of thesamples shown in FIG. 4. FIG. 5A demonstrates undesirable mineralization(red coloring) in the AdBMP2 transduced specimens but not AdNELL1 orAdLacZ specimens. FIG. 5B demonstrates that NELL1 induces significantlymore cartilage mass than AdLacZ controls. FIG. 5C demonstrates thatAdBMP2 significantly increased density (another indicator ofmineralization) in the specimens. These studies quantitativelydemonstrate that although BMP2 induces a larger tissue mass, the inducedmass is largely mineralized and is not consistent with a purelycartilaginous phenotype.

FIG. 6 shows histologic appearance of AdNELL1, AdBMP2, or AdLacZ(control) transduced goat primary chondrocytes 2 weeks afterimplantation/injection into nude mice. Hematoxylin and eosin (H&E)staining (1^(st) row) shows evidence of increased cartilage formation inthe AdNELL1 and AdBMP2 transduced specimens relative to AdLacZ controls.Alcian blue staining which stains cartilage (2^(nd) row) alsodemonstrates increased cartilage formation in the AdNELL1 and AdBMP2transduced specimens relative to AdLacZ controls. Type X collagen (ColX)immunostaining which stains more mature cartilage cells (3^(rd) row)demonstrates increased staining in the AdNELL1 and AdBMP2 transducedspecimens. Collectively, these data indicate that both AdNELL1 andAdBMP2 induce comparable cartilage formation and maturation at 2 weeks.

FIG. 7 shows histologic appearance of AdNELL1, AdBMP2, or AdLacZ(control) transduced goat primary chondrocytes 4 weeks afterimplantation/injection into nude mice. H&E staining (1^(st) row) showssignificant cartilage formation in the AdNELL transduced samples with noevidence of bone formation, while AdBMP2 samples show significant boneformation. A small amount of cartilage formation is seen the AdLacZcontrols. Alcian blue staining (2^(nd) row) also demonstratessignificant cartilage formation in the AdNELL transduced samples with noevidence of bone formation, while AdBMP2 samples show significant boneformation and minimal cartilage formation. A small amount of immaturecartilage formation is seen the AdLacZ controls. Collectively, thesedata indicate that by 4 weeks, AdNELL1 can continue to induce andmaintain a cartilaginous phenotype, while AdBMP2 goes on to form boneand is unable to maintain a cartilaginous phenotype in chondrogeniccells.

FIG. 8 shows immunostaining for bone marker Cbfa1/Runx2 and cartilagemarkers ColX and tenascin in AdNELL1, AdBMP2, or AdLacZ (control)transduced goat primary chondrocytes 4 weeks afterimplantation/injection into nude mice. Tenascin is intimately associatedwith the development of articular cartilage and other permanentcartilages whereas absence or reduced amounts of this matrix proteincharacterize transient cartilages which undergo maturation and arereplaced by bone (Pacifici, M., M. Iwamoto, et al. Tenascin isassociated with articular cartilage development. Dev Dyn 198(2): 123-34,1993). Cbfa1/Runx2 is minimally expressed in cartilaginous AdNELL1 orcontrol AdLacZ transduced samples and moderately expressed in bonyAdBMP2 transduced samples (1^(st) row). ColX is highly expressed andlocalized largely to cells in cartilaginous AdNELL1 samples withoutevidence of bone formation, while ColX is largely associated with theextracelluar matrix rather than cells in the AdBMP2 treated samples(2^(nd) row). Tenascin is highly expressed in AdNELL1 samples andminimally present in AdBMP2 and control AdLacZ samples (3^(rd) row).These studies show NELL1 is able to induce molecules (e.g., tenascin)associated with development of articular cartilage and other permanentcartilages.

FIG. 9 shows immunostaining for endochondral ossification associatedangiogenic growth factor, vascular endothelial growth factor (VEGF), andbone marker osteocalcin (OCN) in AdNELL1, AdBMP2, or AdLacZ (control)transduced goat primary chondrocytes 4 weeks afterimplantation/injection into nude mice. Both VEGF and OCN are notexpressed in cartilaginous AdNELL1 or control AdLacZ transduced samplesand moderately expressed in bony AdBMP2 transduced samples. These datashow that NELL1 does not promote angiogenesis and that NELL1 may inhibitangiogenesis in cartilaginous samples.

FIG. 10 shows the histology of long bone cartilage in NELL-1 overexpression mice. NELL1 is expressed throughout the tibia duringendochondral bone formation including both articular cartilage region(Upper panel) and also the long bone formation region (lower panel).Upper panel demonstrates that NELL1 can modulate and increase cartilagedifferentiation in the articular cartilage region. Accordingly, thesedata show that increased NELL peptide activity directly (e.g., throughaddition of NELL peptides or increased NELL peptide expression) orindirectly (e.g., through addition of NELL peptide enhancers and/or NELLpeptide receptor agonists and/or activators) promotes cartilageformation. In the lower panel, in the long bone shaft region where boneformation originated, increased NELL1 causes cartilage formation andthen hypertrophy and increased bone formation through endochondralossification. Accordingly, these data show that increased NELL peptideactivity directly or indirectly promotes cartilage formation, cartilagehypertrophy and endochondral ossification. The absence of NELL1associates with less differentiated articular chondroblast/chondrocytephenotype and less hypertrophy which is important to prevent articularcartilage replaced by bone. Accordingly, the inhibition of NELL peptideactivity directly (through decreased NELL peptide expression or use ofNELL peptide inhibitors) or indirectly (through NELL peptide receptorantagonists and/or inhibitors) can prevent cartilage hypertrophy andendochondral ossification and promote maintenance of a lessdifferentiated or hypertrophied cartilage phenotype. Overall, these dataare not intended to be limiting, but rather to show that NELL has broadeffects on osteochondroprogenitor cell types and that the exactphenotype induced by NELL depends on a complex interplay between theamount and timing of NELL application, the exact cell type, celldifferentiation state, and the microenvironment.

DEFINITIONS

The term “cartilage” is understood to encompass hyaline, elastic andfibrocartilage and can refer to any cartilaginous component of a mammal.For instance, spinal disc and knee meniscus are fibrocartilaginousstructures that are included in the definition of cartilage.

The terms “polypeptide”, “peptide” and “protein” can be usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms can apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The term “NELL” refers to “NELL1 and NELL2 peptide. A NELL1 peptide is aprotein which can be expressed by the NELL1 gene or cDNA and includesSEQ ID NO: 2, 4, and 6. The NELL1 peptide can include a NELL1 peptidefragment that retains the ability to induce chondrogenic celldifferentiation for cartilage formation. A NELL2 peptide is a proteinwhich can be expressed by the NELL2 gene or cDNA and includes SEQ ID NO:8, 10, 12 and 14. The NELL2 peptide can include NELL2 peptide fragmentsthat retain similar activity to the full NELL2 peptide sequence. Nell-1,Nell-2, etc. intact proteins, completely or partially glycosylated,fragments, deletions, additions, amino acid substitutes, mutations andmodifications that retain the biological characteristics of thenaturally occurring agents. Small molecules containing Nell activedomains and Nell binding sites.

In some embodiments, the term “NELL peptide” can include a fragment of aNELL1 or NELL2 related polypeptide.

In some embodiments, the term “NELL peptide” can include a NELL relatedagent. For example, a NELL peptide related agent can include anypolypeptide with significant homology to a NELL peptide or a fragmentthereof. Significant homology can be a homology of higher than about 50%homology to a NELL peptide, e.g., higher than about 60% homology to aNELL peptide, higher than about 70% homology to a NELL peptide, orhigher than about 80% homology to a NELL peptide.

The NELL peptides can be natural and/or recombinant NELL peptides with anon-mutated wild-type sequence or recombinant NELL peptides with amutated wild-type sequence that still contains significant homology toNELL peptides. In addition, NELL peptides can be derived from, but notlimited to, an organism such as human cells, bacteria, yeast, or insector plant cells. In some embodiments, the term “NELL peptide” includesstructural, functional or conformational equivalents of NELL peptide. Asused herein, a structural equivalent of a NELL peptide refers to aprotein or peptide including a structure equivalent or substantiallysimilar to that of a NELL peptide or of a functional domain of a NELLpeptide. A functional equivalent of a NELL peptide refers to a proteinor peptide having a function equivalent or substantially similar to thatof a NELL peptide or of a functional domain of a NELL peptide. Aconformational equivalent of a NELL peptide refers to a protein orpeptide having a conformation equivalent or substantially similar tothat of a NELL peptide or of a functional domain of a NELL peptide.

In some embodiments, the NELL peptide described herein can be aderivative of the NELL peptide. The term “derivative” as used herein,refers to any chemical or biological compounds or materials derived froma NELL peptide, structural equivalents thereof, or conformationalequivalents thereof. For example, such a derivative can include anypro-drug form, PEGylated form, or any other form of a NELL peptide thatrenders the NELL peptide more stable or to have a better osteo philicityor lipophilicity. In some embodiments, the derivative can be a NELLpeptide attached to poly(ethylene glycol), a poly(amino acid), ahydrocarbyl short chain having C1-C20 carbons, or a biocompatiblepolymer. In some embodiments, the term “derivative” can include a NELLpeptide mimetics. Synthesis of mimetics of a peptide is well document inthe art. The following describes an example of the basic procedure forthe synthesis of a peptide, including a peptide mimetics:

Before the peptide synthesis starts, the amine terminus of the aminoacid (starting material) can protected with FMOC (9-fluoromethylcarbamate) or other protective groups, and a solid support such as aMerrifield resin (free amines) is used as an initiator. Then, step (1)through step (3) reactions are performed and repeated until the desiredpeptide is obtained: (1) a free-amine is reacted with carboxyl terminususing carbodiimide chemistry, (2) the amino acid sequence is purified,and (3) the protecting group, e.g., the FMOC protecting group, isremoved under mildly acidic conditions to yield a free amine. Thepeptide can then be cleaved from the resin to yield a free standingpeptide or peptide mimetics.

In some embodiments, the peptide derivative described herein includes aphysically or chemically modified NELL peptide. Physically modifiedpeptide can be modification by, for example, modification by ionic forcesuch as forming an ionic pair with a counterion, modification byhydrogen bonding, modification by modulation of pH, modulation bysolvent selection, or modification by using different proteinfolding/unfolding procedures, which can involve selection offolding/unfolding temperature, pH, solvent, and duration at differentstage of folding/unfolding.

In some embodiments, the peptide derivative can include a chemicallymodified NELL peptide. For example, a short hydrocarbon group(s) (e.g.methyl or ethyl) can be selectively attached to one or multiple sites onthe NELL peptide molecule to modify the chemical and/or physicalproperties of the peptide. In some embodiments, a mono-, oligo- orpoly(ethylene glycol) (PEG) group(s) can be selectively attached to oneor multiple sites on the NELL peptide molecule to modify the chemicaland/or physical properties of the peptide by commonly known proteinPEGylation procedures (see, e.g., Mok, H., et al., Mol. Ther.,11(1):66-79 (2005)).

The terms “NELL1 cDNA” can refer to SEQ ID NO:1, 3 and 5, and “NELL2cDNA” can refer to SEQ ID NO:7, 9, 11 and 13.

The term “antibody” refers to any antibody that specifically binds to aNELL peptide or a related agent. The term can include various forms ofmodified or altered antibodies, such as an intact immunoglobulin, an Fvfragment containing only the light and heavy chain variable regions, anFv fragment linked by a disulfide bond, a Fab or (Fab)′2 fragmentcontaining the variable regions and parts of the constant regions, asingle-chain antibody and the like. An antibody can include intactmolecules as well as fragments thereof, such as, Fab and F(ab′)^(2′),and/or single-chain antibodies (e.g. scFv) which can bind an epitopicdeterminant. An antibody can be of animal (such as mouse or rat) orhuman origin or can be chimeric or humanized. Antibodies can bepolyclonal or monoclonal antibodies (“mAb's”), such as monoclonalantibodies with specificity for a polypeptide encoded by a NELL1 orNELL2 protein.

The term “capture agent” can refer to molecules that specifically bindother molecules to form a binding complex such as antibody-antigen,lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, and thelike.

The term “specifically binds” can refer to a biomolecule (e.g., protein,nucleic acid, antibody, etc.), refers to a binding reaction which isdeterminative of the presence biomolecule in heterogeneous population ofmolecules (e.g., proteins and other biologics). Thus, under designatedconditions (e.g. immunoassay conditions in the case of an antibody orstringent hybridization conditions in the case of a nucleic acid), thespecified ligand or antibody can bind to its particular “target”molecule and can not bind in a significant amount to other moleculespresent in the sample.

The terms “nucleic acid” or “oligonucleotide” can refer to at least twonucleotides covalently linked together. A nucleic acid of the presentinvention can be single-stranded or double stranded and can containphosphodiester bonds, although in some cases, nucleic acid analogs canbe included that can have alternate backbones, comprising, for example,phosphoramide, phosphorothioate, phosphorodithioate,omethylphophoroamidite linkages, and/or peptide nucleic acid backbonesand linkages. Analog nucleic acids can have positive backbones and/ornon-ribose backbones. Nucleic acids can also include one or morecarbocyclic sugars. Modifications of the ribose-phosphate backbone canbe done to facilitate the addition of additional moieties such aslabels, or to increase the stability and half-life of such molecules inphysiological environments, for example.

The term “specific hybridization” can refer to the binding, duplexing,or hybridizing of a nucleic acid molecule preferentially to a particularnucleotide sequence under stringent conditions, including conditionsunder which a probe can hybridize preferentially to its targetsubsequence, and can hybridize to a lesser extent to other sequences.

The term “inhibitor of NELL peptides” refers to a chemical or biologicalcompound capable of inhibiting the activity of NELL peptides. The termalso includes a chemical or biological compound capable of suppressingthe expression of NELL peptides. Inhibitors of NELL peptides caninteract directly or indirectly with NELL peptide transcripts ortranslational products. As examples, methods of interactions can includebut are not limited to decreased transcription or translation of NELLpeptides, decreased stability of NELL peptide transcripts or proteinproducts, decreased activity of NELL peptide transcripts or proteinproducts, and increased degradation of NELL peptide transcript orprotein products. The term “enhancer of NELL peptides” refers to achemical or biological compound capable of enhancing the activity ofNELL peptides. The term also includes a chemical or biological compoundcapable of enhancing the expression of NELL peptides. As examples,methods of interactions can include but are not limited to increasedtranscription or translation of NELL peptides, increased stability ofNELL peptide transcripts or protein products, increased activity of NELLpeptide transcripts or protein products, and decreased degradation ofNELL peptide transcript or protein products.

The term “modulator of NELL peptide receptors” refers to a chemical orbiological compound capable of facilitating or inhibiting the binding ofNELL peptide receptors to or by NELL peptides or to a chemical orbiological compound capable of modulating NELL peptide receptor activityirrespective of the presence or the absence of NELL peptide. Themodulator that facilitates the binding and/or activation of NELL peptidereceptors to or by NELL peptides is referred to as an “agonist” of thereceptor, and the modulator that inhibits the binding and/or activationof NELL peptide receptors to or by NELL peptides is referred to as an“antagonist” of the receptor. The modulator that facilitates theactivation of NELL peptide receptors irrespective of NELL peptides isreferred to as an “activator” of the receptor, and the modulator thatinhibits activation of NELL peptide receptors irrespective of NELLpeptides is referred to as an “inhibitor” of the receptor.

The term “NELL peptide,” “NELL related agent,” “inhibitor of NELLpeptide” or “modulator of NELL peptide receptor(s)” can also be referredto as an “agent” throughout the specification.

The term “delivery vehicle” refers to any delivery vehicle used in theart of biochemistry. Some examples of common delivery vehicle are anaked DNA type vehicle, an RNA type vehicle, a virus type vehicle. Somefurther examples are e.g., a polymer or a peptide, sustained releasecarriers, synthetic scaffolds, natural scaffolds, allograft or xenograftscaffolds.

The term “mammalian subject” or “mammal” refers to any mammals, examplesof which include human beings and animals such as horse.

Cartilage Formation

Cartilage formation generally proceeds via chondrification process.Chondrification is the process in which cartilage is formed fromcondensed mesenchyme tissue, which differentiates into chondrocytes andbegins secreting the materials that form the matrix. Cartilage canundergo mineralization. Adult hyaline articular cartilage, for example,is progressively mineralized at the junction between cartilage and bone.A mineralization front advances through the base of the hyalinearticular cartilage at a rate dependent on cartilage load and shearstress. Intermittent variations in the rate of advance and mineraldeposition density of the mineralizing front lead to multiple tidemarksin the articular calcified cartilage.

Adult articular calcified cartilage is penetrated by vascular buds, andnew bone produced in the vascular space in a process similar toendochondral ossification at the physis. A cement line demarcatesarticular calcified cartilage from subchondral bone. Two types of growthcan occur in cartilage: appositional and interstitial. Appositionalgrowth results in the increase of the diameter or thickness of thecartilage. The new cells derive from the perichondrium and occur on thesurface of the cartilage model. Interstitial growth results in anincrease of cartilage mass and occurs from within. Chondrocytes undergomitosis within their lacuna but remain imprisoned in the matrix, whichresults in clusters of cells called isogenous groups.

Cartilage can also be formed via endochondral ossification. Themammalian skeleton develops through both endochondral andintramembranous bone formation processes. Embryologically, Duringskeletal development, the establishment of a layer of cartilage at theends of certain bones is intimately linked to the process ofendochondral ossification. The cartilaginous portion of endochondralbone formation involves chondroblast/chondrocyte differentiation,maturation, hypertrophy with or without mineralization depending on thelocation of the cartilage. Non-mineralizing cartilage formation includesbut is not limited to formation of articular cartilage,temporomandibular joint, wrist, knee, and intervertebral discfibrocartilages.

Endochondral ossification or long bone formation is related to boneformation, which permits functional stresses to be sustained duringskeletal growth and is well demonstrated in the development of the longbones. In this process, a small model of the long bone is first formedin solid hyaline cartilage which undergoes mainly appositional growth toform an elongated, dumb-bell shaped mass of cartilage consisting of ashaft (diaphysis) and future articular portions (epiphysis) surroundedby perichondrium (see, e.g., Wheater, P. R. and H. G. Burkitt (1987).Functional histology: a text and colour atlas. Edinburgh; New York,Churchill Livingstone; Beaupre, G. S., S. S. Stevens, et al., J RehabilRes Dev 37(2): 145-51) (2000)).

Within the shaft of the cartilage model then chondrocytes enlargegreatly, resorbing the surrounding cartilage so as to leave only slenderperforated trabeculae of cartilage, matrix. This cartilage matrix thencalcifies and the chondrocytes degenerate leaving large, interconnectingspaces. During this period the perichondrium of the shaft developschondrogenic potential and assumes the role of periosteum. Theperiosteum then lays down a thin layer of bone around the surface of theshaft and primitive mesenchymal cells and blood vessels invade thespaces left within the shaft after degeneration of the chondrocytes. Theprimitive mesenchymal cells differentiate into osteoblasts andblood-forming cells on the surface of the calcified remnants of thecartilage matrix and commence the formation of irregular, woven bone(Wheater and Burkitt, 1987, supra). In the cartilage model described inWheater and Burkitt, 1987, supra, the ends of the original cartilagemodel have then become separated by a large site of primary ossificationin the shaft. The cartilaginous ends of the model, however, continue togrow in diameter. Meanwhile, the cartilage at the ends of the shaftcontinues to undergo regressive changes followed by ossification so thatthe developing bone now consists of an elongated, bony diaphyseal shaftwith a semilunar cartilage epiphysis at each end. The interface betweenthe shaft and each epiphysis constitutes a growth or epiphyseal plate.Within the growth plate, the cartilage proliferates continuously,resulting in progressive elongation of the bone. At the diaphysealaspect of each growth plate, the chondrocytes mature and then die, thedegenerating zone of cartilage being replaced by bone. Thus the bonydiaphysis lengthens and the growth plates are pushed further and furtherapart. On reaching maturity, hormonal changes inhibit further cartilageproliferation and the growth plates are replaced by bone causing fusionof the diaphysis and epiphysis (Wheater and Burkitt, 1987, supra). Inthe meantime, in the center of the mass of cartilage of each developingepiphysis, regressive changes and bone formation similar to that in thediaphyseal cartilage occur along with appositional growth of cartilageover the whole external surface of the epiphysis. This conversion ofcentral epiphyseal cartilage to bone is known as secondary ossification.A thin zone of hyaline cartilage always remains at the surface as thearticular cartilage (Wheater and Burkitt, 1987, supra).

Thus, endochondral bone formation and growth is achieved in part by theproliferation and maturation of cartilage cells (chondroblasts,chondrocytes) with or without cartilage cell mineralization. Cartilageformation or regeneration can be achieved by controlling cartilage cellmineralization. Without being bound by a particular theory, cartilagecell mineralization can be controlled by controlling factors such as: a)location, b) cell type, c) cell differentiation state, d)microenvironment, and e) bioimechanical forces. For example, themineralization of a cartilage cell can be controlled by placing thecartilage cell near an epiphyseal growth plate in which mineralizationgenerally occurs or near an articular surface in which mineralizationgenerally does not occur. It is known in the art that chondrocytehypertrophy and up-regulated matrix calcification are dissociable states(see, e.g., Johnson, van Etten et al. 2003) (see, e.g., Johnson, K. A.,D., et al., J Biol Chem 278(21):18824-32 (2003)). For example, theformation of endochondral bone can be evaluated by chondroblasthypertrophy as viewed by an increase in hypertrophic and apoptoticchondroblasts, elucidated by TUNEL staining. In another example, theformation of cartilage can be evaluated also by chondroblast hypertrophywithout necessarily apoptosis or mineralization.

Cartilage Regeneration

Cartilage contains a significant amount of water. For instance,articular cartilage is comprised of mostly water (60-80 wt %) and theremaining ECM comprises mostly type II collagen (50-90% dry mass) andproteoglycans (5-10%). Other collagens and minor ECM molecules have beenidentified in small quantities. It is organization of the ECM intodistinct zones, and the interaction between water and the ECM in thevarious zones that provide the toughness that is required for theabsorption and transmission of biomechanical forces across joints, andsimultaneously the frictionless articulating surfaces that are neededfor joint motion. Stresses as high as 4 and 20 MPa have been reported inhuman hip joints during routine walking and jumping, respectively! Asamazing as the articular cartilage is, it exhibits unfortunately minimalcapacity for repair. Over 20 million Americans suffer fromosteoarthritis and degenerative joint diseases with an associated annualhealthcare burden of over $60 billion. A wide array of scaffolds,cytokines, and growth factors have been investigated for cartilagetissue engineering (see, e.g., Frenkel, S. R., et al., Ann. Biomed. Eng.32:26-34 (2004); Tuli, R., et al., Arthritis Res. Ther. 5:235-238(2003); and Ashammakhi, N. and Reis, R L. Topics in Tissue Engineering,Vol. 2, 2005). The role of static vs. dynamic compression, shear stress,hydrostatic pressure, fluid flow, electrical streaming potentials,bioreactors, and complex loading on chondrocyte biological response andtissue remodeling have been investigated extensively and themechanotransduction pathways reviewed Ashammakhi, N. and Reis, R L.Topics in Tissue Engineering, Vol. 2, 2005) (see FIGS. 7A-D therein)

Accordingly, in a further aspect of the present invention, thecomposition provided herein includes at least a NELL peptide or anagonist of the receptor of NELL peptides in an amount effective forinducing chondroblast and chondrocyte to form cartilage. NELL proteins,peptides, DNA, RNA, and NELL agonists, and antagonist inhibitors can beused alone or in conjunction with scaffolds with and without cells, withor without mechanical stimulation, in the presence or absence ofadditional growth factors. For example, in one embodiment, thecomposition can be effective in regenerating or repairing or augmentingcartilage in intervertebral disc, temporomandibular disc, knee and wristfibrocartilage, and articular surfaces. In another embodiment, thecomposition can be effective in forming cartilage via ex vivo genetherapy and protein application to cells with or without scaffold intissue engineering.

Depending on the delivery method and the local environment, acomposition including a NELL peptide (e.g., a NELL1 peptide) can be usedto induce an chondrogenic cell, as such as a chondrocyte orchondroblast, to differentiate and form cartilage only. For example, inan articular cartilage defect, the composition described herein caninduce an chondrogenic cell such as chondrocyte/blast to form cartilageonly. The composition can be applied to the defected cartilage area as ascaffold/carrier. In some embodiments, the composition can optionallyinclude cells (stem cells, chondroblast etc). In some embodiments, thecomposition can be applied as gene therapy.

In some embodiments, as used herein, the cells can be, e.g.,differentiated chondrocytes; differentiated cells (e.g. skeletal musclecells, fibroblasts) that are de-differentiated after implantation, orprior to implantation; adult stem cells that are differentiated afterimplantation, or prior to implantation; embryonic stem cells that aredifferentiated after implantation, or prior to implantation; human;modified by nucleic acid, protein, small molecules, siRNA, antibodies.

In some yet embodiments, the composition can be used in cartilage tissueengineering. For example, when chondroblasts are cultured on an“oscillating”, intermittent stress tension environment, NELL1 peptidecan include the chondroblast cells to differentiate and form cartilage.In these embodiments, the duration of application of the oscillatingstress also plays an important role. For example, if the oscillatingforce is applied continuously, the composition having a NELL1 peptidecan induce endochondral bone formation. Therefore, in the application ofthe oscillating stress shall be intermittently such that thedifferentiation of an chondrogenic cell (e.g., chondrocyte/blast) canstop at the cartilage stage and thus prevent the cell fromdifferentiating into endochondral bone formation.

Therefore, in some embodiments, the composition described herein can beused to regenerate/repair cartilage, e.g., for disc repair in articularcartilage and intervertebral disc.

Other exemplary cartilage conditions that can be treated, prevented, orameliorated by a composition disclosed herein include, but are notlimited to, chondrocalcinosis, osteoarthritis, and/or other diseasescharacterized by pathological cartilage degeneration.

In one embodiment, a method of increasing endochondral bone formationcan include increasing the concentration of a NELL1 gene product in aregion where bone formation is desired; optionally applying a secondagent to the region where bone formation is desired and at leastinducing hypertrophy of chondroblast in the region where bone formationis desired.

The method can include increasing the concentration of a NELL1 geneproduct by applying a NELL1 peptide to the location where bone formationis desired, and the NELL1 peptide can be selected from the groupcomprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6, or any portion ofthe NELL peptide which is effective in increasing endochondral boneformation, which involves both cartilage and bone.

The second agent can include, but is not limited to TGF-beta, BMP2,BMP4, BMP7, bFGF, insulin like growth factor (IGF), Sox9, collagen,chondrogenic cells, bone, bone matrix, tendon matrix, ligament matrix.The second agent can be selected to have a complimentary or synergisticeffect with NELL1 in inducing endochondral bone formation. Other agentsare described below.

Inhibition of Angiogenesis and Cartilage Formation/Regeneration

As specified in Shukunami et al., cartilage forms a template for most ofthe bony skeleton in embryonic development (Shukunami, C., Y. Oshima, etal., Biochem Biophys Res Commun 333(2): 299-307) (2005)). Cartilage isnot directly converted to bone but is gradually replaced through theactions of osteoclasts and osteoblasts, which are brought to theossification center of cartilage with vascular invasion (endochondralbone formation). Thus, the vascular invasion of cartilage can be crucialfor bone formation at an appropriate stage of development. Cartilageacquires an anti-angiogenic nature upon chondrogenesis and quickly losesit, as chondrocytes mature to become hypertrophic and calcified prior tovascular invasion, suggesting that cartilage undergoes a dynamicswitching of the anti-angiogenic phenotype. Undoubtedly pro-angiogenicfactors act as a driving force for vascular invasion into tissues.VEGF-A is a key regulator of angiogenesis during endochondral boneformation: VEGF-A is expressed in hypertrophic cartilage, but not inresting or proliferating cartilage.

Matrix metalloproteinases (MMPs) can influence bone development, whichinvolves matrix-remodeling during vascular invasion (e.g., MMP-9,MMP-13, MMP-14). In mice lacking MMP-9, vascular invasion and subsequentossification were delayed, causing progressive lengthening of the growthplate. The delay in ossification appeared to be secondary to adiminished vascular invasion of cartilage probably becauseMMP-9-deficient hypertrophic cartilage fails to release normal levels ofpro-angiogenic activity to stimulate vessel formation and to recruitosteo/chondroclasts. Targeted inactivation of MMP-14 (membrane type 1MMP: MT1-MMP) causes severe defects in both endochondral andintramembranous bone formation in mice. These results indicate that MMPsplay a regulatory role in angiogenic switching of the cartilagephenotype. Thus, an important part of cartilage formation andregeneration can involve differential regulation of pro-angiogenicfactors such as MMP-9, MMP-13, MMP-14, and VEGF and anti-angiogenicfactors such as chondromodulin-I (ChM-I), thrombospondin (TSP)-1, TSP-2,tissue inhibitor of metalloproteinase (TIMP)-2, TIMP-3. Specifically,pro-angiogenic factors can be relatively more prominent in areas ofcartilage undergoing ossification, and anti-angiogenic factors may berelatively more prominent in areas of cartilage not undergoingossification. These results also indicate that the transcription factorCbfa1/Runx2 can be involved in the control of angiogenic switching incartilage: Cbfa1/Runx2 null mice are defective in hypertrophic cartilagedifferentiation, vascular invasion of cartilage rudiments, and VEGFexpression, and exhibit a sustained expression of the ChM-I gene. InCbfa1/Runx2 null mice expressing the Cbfa1/Runx2 transgene in nonhypertrophic chondrocytes, vascular invasion, and cartilage remodelingwas restored with the upregulation of VEGF and concomitantdownregulation of ChM-I gene expression.

Without being bound by a particular theory, NELL1 can have a role in theangiogenic switching in cartilage, since NELL1 is a direct downstreameffector of Cbfa1/Runx2 effects. In addition without being bound by aparticular theory, NELL1's role in cartilage formation can also relateto potential anti-angiogenic effects of NELL1——as NELL1 also contains aNH₂-terminal thrombospondin-like module.

Other Agents

In one embodiment, the composition for cartilage formation andregeneration described herein can include one or more other agents. Suchagents can be chondroprotective agents, anti-pain and/oranti-inflammatory agents, growth factors, anti-angiogenic agents, orcombinations thereof.

The chondroprotective agents can be, for example, (1) antagonists ofreceptors for the interleukin-1 family of proteins, including, forexample, IL-1.beta., IL-17 and IL-18; (2) antagonists of the tumornecrosis factor (TNF) receptor family, including, for example, TNF-R1;(3) agonists for interleukin 4, 10 and 13 receptors; (4) agonists forthe TGF-.beta. receptor superfamily, including, for example, BMP-2,BMP-4 and BMP-7; (5) inhibitors of COX-2; (6) inhibitors of the MAPkinase family, including, for example, p38 MAP kinase; (7) inhibitors ofthe matrix metalloproteinases (MMP) family of proteins, including, forexample, MMP-3 and MMP-9; (8) inhibitors of the NF-.kappa.B family ofproteins, including, for example, the p50/p65 dimer complex withI.kappa.B; (9) inhibitors of the nitric oxide synthase (NOS) family,including, for example, iNOS; (10) agonists and antagonists of integrinreceptors, including, for example, agonists of α_(v)β₃ integrin; (11)inhibitors of the protein kinase C (PKC) family; (12) inhibitors of theprotein tyrosine kinase family, including, for example, the srcsubfamily; (13) modulators of protein tyrosine phosphatases; and (14)inhibitors of protein src homology 2 (SH2) domains. Additionalchondroprotective agents include other growth factors, such as by way ofexample insulin-like growth factors (e.g., IGF-1) and fibroblast growthfactors (e.g., bFGF). Other chondroprotective agents are described inU.S. Pat. No. 7,067,144, the teachings of which are incorporated hereinby reference. These chondroprotective agents can be used alone or incombination along with a NELL peptide or related agent. In someembodiments, the composition described herein can specifically excludeany of the above described chondroprotective agents.

The anti-pain and/or anti-inflammatory agents can be, e.g., (1)serotonin receptor antagonists; (2) serotonin receptor agonists; (3)histamine receptor antagonists; (4) bradykinin receptor antagonists; (5)kallikrein inhibitors; (6) tachykinin receptor antagonists, includingneurokinin.sub.1 and neurokinin.sub.2 receptor subtype antagonists; (7)calcitonin gene-related peptide (CGRP) receptor antagonists; (8)interleukin receptor antagonists; (9) inhibitors of enzymes active inthe synthetic pathway for arachidonic acid metabolites, including (a)phospholipase inhibitors, including PLA.sub.2 isoform inhibitors and PLCisoform inhibitors, (b) cyclooxygenase inhibitors, and (c) lipooxygenaseinhibitors; (10) prostanoid receptor antagonists including eicosanoidEP-1 and EP-4 receptor subtype antagonists and thromboxane receptorsubtype antagonists; (11) leukotriene receptor antagonists includingleukotriene B.sub.4 receptor subtype antagonists and leukotriene D.sub.4receptor subtype antagonists; (12) opioid receptor agonists, includingμ-opioid, δ-opioid, and .kappa.-opioid receptor subtype agonists; (13)purinoceptor antagonists including P₂X receptor antagonists and P₂Yreceptor antagonists; and (14) calcium channel antagonists. Each of theabove agents functions either as an anti-inflammatory agent and/or as ananti-nociceptive (i.e., anti-pain or analgesic) agent. The selection ofagents from these classes of compounds is tailored for the particularapplication. These anti-pain and/or anti-inflammatory agents can be usedalone or in combination along with a NELL peptide or related agent. Insome embodiments, the composition described herein can specificallyexclude any of the above described anti-pain and/or anti-inflammatoryagents.

The growth factors can be, e.g., FGF-2, FGF-5, IGF-1, TGF-.beta., BMP-2,BMP-7, PDGF, VEGF, OP1, OP2, OP3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP9,BMP10, BMP11, BMP12, BNP15, BMP16, DPP, Vgl, 60A protein, GDF-1, GDF3,GDF5, GDF6, GDF7, GDF8, GDF9, GDF10 and GDF11. Some other growth factorsare described in U.S. Pat. Nos. 7,067,123, and 7,041,641, the teachingsof which are incorporated herein by reference. These growth factors canbe used alone or in combination along with a NELL peptide or relatedagent. In some embodiments, the composition described herein canspecifically exclude any of the above described growth factors.

The anti-angiogenic agents can be, e.g., anti-angiogenic factors,including for example Anti-Invasive Factor, retinoic acids and theirderivatives, paclitaxel including analogues and derivatives thereof,Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor ofMetalloproteinase-2, Plasminogen Activator Inhibitor-1 and PlasminogenActivator Inhibitor-2, and lighter “d group” transition metals.Similarly, a wide variety of polymeric carriers may be utilized,representative examples of which include poly(ethylene-vinyl acetate)(40% cross-linked), poly(D,L-lactic acid) oligomers and polymers,poly(L-lactic acid) oligomers and polymers, poly(glycolic acid),copolymers of lactic acid and glycolic acid, poly(caprolactone),poly(valerolactone), poly(anhydrides), copolymers of poly(caprolactone)or poly(lactic acid) with polyethylene glycol, and blends thereof.Within certain preferred embodiments, the compositions comprise acompound which disrupts microtubule function, such as, for example,paclitaxel, estramustine, colchicine, methotrexate, curacin-A,epothilone, vinblastine or tBCEV. Within other preferred embodiments,the compositions comprise a polymeric carrier and a lighter d grouptransition metal (e.g., a vanadium species, molybdenum species, tungstenspecies, titanium species, niobium species or tantalum species) whichinhibits the formation of new blood vessels (as specified in USP20060240113), inhibitors of VEGF (as specified in USP 20060241084),other inhibitors of angiogenesis (as specified in USP 20060235034,7,122,635), chondromodulin-I or tenomodulin (Shukunami, et al., 2005,supra), or other endogenous or exogenous anti-angiogenic factors wellknown to those in the art.

Formulations

The composition described herein can be formulated into any desiredformulation. The composition can include materials and carriers toeffect a desired formulation. For example, the composition can includean injectable or moldable material that can set within a pre-definedperiod of placement. Such a pre-defined period can be, e.g., 10 minutes,30 minutes, one hour, two hours, etc.

In some embodiments, the composition can include a chemical gel thatincludes primary bonds formed due to changes in pH, ionic environment,and solvent concentration. Examples of such chemical gels can be, butare not limited to, polysaccharides such as chitosan, chitosan plusionic salts such as beta-glycerophosphates, aginates plus Ba2+, Sr2+,Ca2+, Mg2+, collagen, fibrin, plasma or combinations thereof.

In some embodiments, the composition can include a physical gel thatinclude secondary bonds formed due to temperature changes. Examples ofsuch physical gels can be, but are not limited to, alginate,poly(ethylene glycol)-poly(lactic acid-co-glycolic acid)-poly(ethyleneglycol) (PEG-PLGA-PEG) tri-block copolymers, agarose, and celluloses. Insome embodiments, physical gels that can be used in the compositiondescribed herein can include physical gels that are liquid under highshear but gels to solid at low shear. Examples of such physical gelsinclude, but are not limited to, hyaluronic acid, or polyethyleneoxides. The physical gels can have pre-formed materials with pre-defineddimensions and shape.

In some embodiments, the composition described herein can include amaterial that degrade or release active agents in response to astimulus. Some examples of such stimuli are mechanical stimuli, light,temperature changes, pH changes, change of ionic strength, orelectromagnetic field. Such materials are know in the art some examplesof such materials are chitosan, alginates, pluronics, methyl cellulose,hyaluronic acids, and polyethylene oxides. Other examples are describedby Brandl F, Sommer F, Goepferich A. “Rational design of hydrogels fortissue engineering: Impact of physical factors on cell behavior” inBiomaterials. Epub Sep. 29, 2006.

In some embodiments, the composition described herein can include a gelcontaining any of hydroxyapatites, apatites, tricalcium phostphates,calcium phosphates, bioactive glass, human allograft bone and cartilage,bovine bone and cartilage, or their mixtures thereof.

In some embodiments, the composition described herein including any ofthe gels described above can further include a crosslinker to furthertailor degradation kinetics and controlled release. Alternatively, insome embodiments, the composition described herein can include aninterpenetrating phase composite or interpenetrating network (IPN) thatincludes any of the above described gels. Some examples of thecrosslinker includes, but are not limited to, common crosslinking agents(polyalkylene oxide, ethylene dimethacrylate,N,N′-methylenebisacrylamide, methylenebis(4-phenyl isocyanate), ethylenedimethacrylate, divinylbenzene,allyl methacrylate, carbodiimidazole,sulfonyl chloride, chlorocarbonates, n-hydroxysuccinimide ester,succinimidyl ester, epoxides, aryl halides, sulfasuccinimidyl esters,and maleimides); PEG based crosslinkers (e.g. MAL-dPEGx-NHS-esters,MAL-dPEGx acid, Bis-MAL-dPEGx, etc.) and photo/light activatedcrosslinkers, N-hydroxysuccinimide-based crosslinkers, dilysine,trilysine, and tetralysine.

The composition described herein can include a carrier. The carrier canbe a polymeric carrier or non-polymeric carrier. In some embodiments,the carrier can be biodegradable, such as degradable by enzymatic orhydrolytic mechanisms. Examples of carriers include, but are not limitedto synthetic absorbable polymers such as such as but not limited topoly(α-hydroxy acids) such as poly(L-lactide) (PLLA), poly(D, L-lactide)(PDLLA), polyglycolide (PGA), poly(lactide-co-glycolide (PLGA),poly(-caprolactone), poly(trimethylene carbonate), poly(p-dioxanone),poly(-caprolactone-co-glycolide), poly (glycolide-co-trimethylenecarbonate) poly(D, L-lactide-co-trimethylene carbonate), polyarylates,polyhydroxybutyrate (PHB), polyanhydrides, poly(anhydride-co-imide),propylene-co-fumarates, polylactones, polyesters, polycarbonates,polyanionic polymers, polyanhydrides, polyester-amides,poly(amino-acids), homopolypeptides, poly(phosphazenes),poly(glaxanone), polysaccharides, and poly(orthoesters), polyglactin,polyglactic acid, polyaldonic acid, polyacrylic acids, polyalkanoates;copolymers and admixtures thereof, and any derivatives andmodifications. See for example, U.S. Pat. No. 4,563,489, and PCT Int.Appl. No. WO/03024316, herein incorporated by reference. Other examplesof carriers include cellulosic polymers such as, but not limited toalkylcellulose, hydroxyalkylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropyl-methylcellulose, carboxymethylcellulose, and theircationic salts. Other examples of carriers include synthetic and naturalbioceramics such as, but not limited to calcium carbonates, calciumphosphates, apatites, bioactive glass materials, and coral-derivedapatites. See for example U.S. Patent Application 2002187104; PCT Int.Appl. WO/9731661; and PCT Int. Appl. WO/0071083, herein incorporated byreference.

In one embodiment, the carrier can further be coated by compositions,including bioglass and or apatites derived from sol-gel techniques, orfrom immersion techniques such as, but not limited to simulated bodyfluids with calcium and phosphate concentrations ranging from about 1.5to 7-fold the natural serum concentration and adjusted by various meansto solutions with pH range of about 2.8-7.8 at temperature from about15-65 degrees C. See, for example, U.S. Pat. Nos. 6,426,114 and6,013,591; and PCT Int. Appl. WO/9117965 herein incorporated byreference.

Other examples of carriers include, collagen (e.g. Collastat, Helistatcollagen sponges), hyaluronan, fibrin, chitosan, alginate, and gelatin.See for example, PCT Int. Appls. WO/9505846; WO/02085422, hereinincorporated by reference.

In one embodiment, the carrier can include heparin-binding agents;including but not limited to heparin-like polymers e.g. dextran sulfate,chondroitin sulfate, heparin sulfate, fucan, alginate, or theirderivatives; and peptide fragments with amino acid modifications toincrease heparin affinity. See for example, Journal of BiologicalChemistry (2003), 278(44), p. 43229-43235, herein incorporated byreference.

In one embodiment, the composition can be in the form of a liquid, solidor gel. In one embodiment, the substrate can include a carrier that isin the form of a flowable gel. The gel can be selected so as to beinjectable, such as via a syringe at the site where cartilage formationis desired. The gel can be a chemical gel which can be a chemical gelformed by primary bonds, and controlled by pH, ionic groups, and/orsolvent concentration. The gel can also be a physical gel which can beformed by secondary bonds and controlled by temperature and viscosity.Examples of gels include, but are not limited to, pluronics, gelatin,hyaluronan, collagen, polylactide-polyethylene glycol solutions andconjugates, chitosan, chitosan & b-glycerophosphate (BST-gel),alginates, agarose, hydroxypropyl cellulose, methyl cellulose,polyethylene oxide, polylactides/glycolides in N-methyl-2-pyrrolidone.See for example, Anatomical Record (2001), 263(4), 342-349, hereinincorporated by reference.

In one embodiment, the carrier can be photopolymerizable, such as byelectromagnetic radiation with wavelength of at least about 250 nm.Example of photopolymerizable polymers include polyethylene (PEG)acrylate derivatives, PEG methacrylate derivatives, propylenefumarate-co-ethylene glycol, polyvinyl alcohol derivatives,PEG-co-poly(-hydroxy acid) diacrylate macromers, and modifiedpolysaccharides such as hyaluronic acid derivatives and dextranmethacrylate. See for example, U.S. Pat. No. 5,410,016, hereinincorporated by reference.

In one embodiment, the substrate can include a carrier that istemperature sensitive. Examples include carriers made fromN-isopropylacrylamide (NiPAM), or modified NiPAM with lowered lowercritical solution temperature (LCST) and enhanced peptide (e.g. NELL1)binding by incorporation of ethyl methacrylate andN-acryloxysuccinimide; or alkyl methacrylates such as butylmethacrylate,hexylmethacrylate and dodecylmethacrylate. PCT Int. Appl. WO/2001070288;U.S. Pat. No. 5,124,151 herein incorporated by reference. In oneembodiment, where the carrier can have a surface that is decoratedand/or immobilized with cell adhesion molecules, adhesion peptides, andadhesion peptide analogs which can promote cell-matrix attachment viareceptor mediated mechanisms, and/or molecular moieties which canpromote adhesion via non-receptor mediated mechanisms binding such as,but not limited to polycationic polyamino-acid-peptides (e.g.poly-lysine), polyanionic polyamino-acid-peptides, Mefp-class adhesivemolecules and other DOPA-rich peptides (e.g. poly-lysine-DOPA),polysaccharides, and proteoglycans. See for example, PCT Int. Appl.WO/2004005421; WO/2003008376; WO/9734016, herein incorporated byreference.

In one embodiment, the carrier can include various naturally occurringmatrices or their components such as devitalized cartilage matrix,demineralized bone matrix, or other components derived from allograft,xenograft, or any other naturally occurring material derived fromMonera, Protista, Fungi, Plantae, or Animalia kingdoms.

In one embodiment, the carrier can include comprised of sequesteringagents such as, but not limited to, collagen, gelatin, hyaluronic acid,alginate, poly(ethylene glycol), alkylcellulose (includinghydroxyalkylcellulose), including methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropyl-methylcellulose, and carboxymethylcellulose, blood,fibrin, polyoxyethylene oxide, calcium sulfate hemihydrate, apatites,carboxyvinyl polymer, and poly(vinyl alcohol). See for example, U.S.Pat. No. 6,620,406, herein incorporated by reference.

In one embodiment, the carrier can include surfactants to promote NELL1stability and/or distribution within the carrier materials such as, butnot limited to polyoxyester (e.g. polysorbate 80, polysorbate 20 orPluronic F-68).

In one embodiment, the carrier can include buffering agents such as, butnot limited to glycine, glutamic acid hydrochloride, sodium chloride,guanidine, heparin, glutamic acid hydrochloride, acetic acid, succinicacid, polysorbate, dextran sulfate, sucrose, and amino acids. See forexample, U.S. Pat. No. 5,385,887, herein incorporated by reference. Inone embodiment, the carrier can include a combination of materials suchas those listed above. By way of example, the carrier can a bePLGA/collagen carrier membrane. The membrane can be soaked in a solutionincluding NELL1 peptide.

In one embodiment, an implant for use in the human body can include asubstrate including NELL1 in an amount sufficient to induce cartilageformation or repair proximate to the implant.

In one embodiment, an implant for use in the human body can include asubstrate having a surface including NELL1 in an amount sufficient toinduce cartilage formation or repair proximate to the implant.

In one embodiment, an implant for use in the human body can include asubstrate having a surface including chondrogenic cells, and NELL1 in anamount sufficient to induce cartilage formation or repair. In oneembodiment, the implant can be seeded with cells, including but notlimited to autologous cells, chondrogenic or osteoblastic cells, cellsexpressing NELL1 or another chondrogenic molecule.

An implant can include a substrate formed into the shape of a mesh, pin,screw, plate, or prosthetic joint. By way of example, a substrate can bein a form of a dental or orthopedic implant, and NELL1 can be used toenhance integration in bone in proximity to the implant. An implant caninclude a substrate that is resorbable, such as a substrate includingcollagen.

The NELL1 peptide can be combined with a acceptable carrier to form apharmacological composition. Acceptable carriers can contain aphysiologically acceptable compound that acts, for example, to stabilizethe composition or to increase or decrease the absorption of the agent.Physiologically acceptable compounds can include, for example,carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins, compositions that reduce the clearance or hydrolysis of theanti-mitotic agents, or excipients or other stabilizers and/or buffers.

Other physiologically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives which areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art wouldappreciate that the choice of a carrier, including a physiologicallyacceptable compound depends, for example, on the route ofadministration.

The compositions can be administered in a variety of unit dosage formsdepending upon the method of administration. For example, unit dosageforms suitable can include powder, or injectable or moldable pastes orsuspension.

The compositions of this invention can comprise a solution of the NELL1peptide dissolved in a pharmaceutically acceptable carrier, such as anaqueous carrier for water-soluble peptides. A variety of carriers can beused, e.g., buffered saline and the like. These solutions are sterileand generally free of undesirable matter. These compositions can besterilized by conventional, well known sterilization techniques. Thecompositions can contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like.

The concentration of NELL1 peptide in these formulations can varywidely, and will be selected primarily based on fluid volumes,viscosities, body weight and the like in accordance with the particularmode of administration selected and the patient's needs.

The dosage regimen will be determined by the clinical indication beingaddressed, as well as by various patient variables (e.g. weight, age,sex) and clinical presentation (e.g. extent of injury, site of injury,etc.).

However, a therapeutically effective dose of a NELL1 peptide or agentuseful in this invention is one which has a positive clinical effect ona patient or desired effect in cells as measured by the ability of theagent to enhance chondrogenic differentiation for cartilage formation orrepair, as described above. The therapeutically effective dose of eachpeptide or agent can be modulated to achieve the desired clinicaleffect, while minimizing negative side effects. The dosage of thepeptide or agent can be selected for an individual patient dependingupon the route of administration, severity of the disease, age andweight of the patient, other medications the patient is taking and otherfactors normally considered by an attending physician, when determiningan individual regimen and dose level appropriate for a particularpatient.

Device

The composition can be formulated into an injectable or implantabledevice in any desired form. Some exemplary devices can be forintervertebral disc nucleus replacement, knee meniscus replacement,wrist triangular fibrocartilage replacement, temporomandibular jointreplacement, articular cartilage replacement and can consist of, porousscaffold with preformed shape and attachment features to anchor tounderlying bone; viscous gel with preformed shape that can be re-shapedby manual manipulation and the cured to new shape by the application oflight; or low viscosity liquid that can polymerize in situ. For example,the composition can be formulated into a single mixture (or a simplemixture) for cartilage formation.

In some embodiments, the composition can be formulated into a singledevice containing specifically designed layers that are tissue-specific,e.g. it may be desirable to have a bone layer to anchor to the hardtissues, and then a cartilage layer immediately adjacent to the bonelayer.

In some embodiments, the composition can be formulated into a singlemixture allowing multiple tissues formation and self-assembly, such aspolymers or monomers with amphiphilic functional groups canself-assemble into macroscopic structures.

In some embodiments, where a device including a composition describedherein having a cell(s), the device can be subjected to pre-implantationstimulation. For example, the device can be placed in a mechanicalbioreactor with controlled mechanical stimulation (frequency, dutycycle, amplitude, etc.); Frequency in the range of 0.01 Hz to 10,000 Hz,duty cycle above 10%; and amplitude in the range of 0.1-100% strain havereported enhanced cellular function. In some embodiments, the devicedescribed herein can be placed in a mechanical bioreactor withcontrolled microfluidic flow and shear stresses, which arise when atleast one flow path or channel has one dimension less than 1 mm. In someembodiments, a device described herein can be implanted in a human beingvia direct implantation immediately following cell harvesting.

In some embodiments, the composition provided herein can form any of thefollowing examples of devices, which illustrate, but shall not beconstrued to limit the claimed invention:

An injectable/implantable device containing NELL protein (with orwithout cells) that can be directly injected/implanted into spinal discsto promote cartilage formation;

A disc nucleus replacement device impregnated with NELL that is designedto replace the inner portion of the vertebral disc (the nucleus) or boththe inner and outer portion of the disc;

An injectable/implantable device containing NELL (with or without cells)that can be directly injected into the various joint spaces (e.g., knee,temporomandibularjoint, wrist) or implanted arthroscopically or openlyinto various joint spaces;

An injectable/implantable device containing NELL nucleic acids (with orwithout delivery vehicle such as a virus) (with or without cells) thatcan be directly injected/implanted into spinal discs to promotecartilage formation;

A disc nucleus replacement device impregnated with NELL nucleic acids(with or without delivery vehicle such as a virus) that is designed toreplace the inner portion of the vertebral disc (the nucleus) or boththe inner and outer portion of the disc;

An injectable/implantable device containing NELL nucleic acids (with orwithout delivery vehicle such as a virus) (with or without cells) thatcan be directly injected into the various joint spaces (e.g., knee,temporomandibular joint, wrist) or implanted arthroscopically or openlyinto various joint spaces;

An injectable/implantable device containing NELL protein (with orwithout cells) and other factors that can be directly injected/implantedinto spinal discs to promote cartilage formation;

A disc nucleus replacement device impregnated with NELL and otherfactors that is designed to replace the inner portion of the vertebraldisc (the nucleus) or both the inner and outer portion of the disc;

An injectable/implantable device containing NELL and other factors (withor without cells) that can be directly injected into the various jointspaces (e.g., knee, temporomandibular joint, wrist) or implantedarthroscopically or openly into various joint spaces;

An injectable/implantable device containing NELL nucleic acids and otherfactors (with or without delivery vehicle such as a virus) (with orwithout cells) that can be directly injected/implanted into spinal discsto promote cartilage formation;

A disc nucleus replacement device impregnated with NELL nucleic acids(with or without delivery vehicle such as a virus) that is designed toreplace the inner portion of the vertebral disc (the nucleus) or boththe inner and outer portion of the disc;

An injectable/implantable device containing NELL nucleic acids (with orwithout delivery vehicle such as a virus) (with or without cells) thatcan be directly injected into the various joint spaces (e.g., knee,temporomandibular joint, wrist) or implanted arthroscopically or openlyinto various joint spaces.

Dosages

Dosages of NELL peptides and other agents can be determined according tomethods known in the art based on type of agent, the disease, and otherfactors such as age and gender.

In one embodiment, the dosage of NELL peptide for cartilage formation orrepair generally ranges from 0.001 pg/mm² to 1 pg/mm², or morepreferably from 0.001 ng/mm² to 1 ng/mm², or more preferably from 0.001μg/mm² to 1 μg/mm², or more preferably from 0.001 mg/mm² to 1 mg/mm², ormore preferably from 0.001 g/mm² to 1 g/mm², with or without aparticular carrier or scaffold. In another embodiment, the dosage ofNELL peptide for cartilage formation or repair generally ranges from0.001 pg/ml to 1 pg/ml, or more preferably from 0.001 ng/ml to 1 ng/ml,or more preferably from 0.001 μg/ml to 1 μg/ml, or more preferably from0.001 mg/ml to 1 mg/ml, or more preferably from 0.001 g/ml to 100 g/ml,with or without a particular carrier or scaffold. In yet anotherembodiment, the dosage of NELL peptide for cartilage formation or repairgenerally ranges from 0.001 pg/kg to 1 pg/kg, or more preferably from0.001 ng/kg to 1 ng/kg, or more preferably from 0.001 gg/kg to 1 gg/kg,or more preferably from 0.001 mg/kg to 1 mg/kg, or more preferably from0.001 gm/kg to 1 gm/kg, more preferably from 0.001 kg/kg to 1 kg/kg withor without a particular carrier or scaffold. Furthermore, it isunderstood that all dosages can be continuously given or divided intodosages given per a given timeframe. Examples of timeframes include butare not limited to every 1 hour, 2 hour, 4 hour, 6 hour, 8 hour, 12hour, 24 hour, 48 hour, or 72 hour, or every week, 2 weeks, 4 weeks, orevery month, 2 months, 4 months, and so forth.

However, because NELL peptides can have effects on in vitro osteoblastapoptosis (Zhang, X., et al., J Bone Miner Res, 2003. 18(12): p.2126-34), NELL dosages (e.g., NELL1 dosages) that are significantlyabove an optimal range can not increase cartilage formation or repair.Accordingly, even more preferable dosages of NELL peptide shall not besignificantly above the optimal dosage range. The even more preferableoptimal dosage ranges of NELL peptides can vary according to factorssuch as the type, the age, the location, and the gender of a mammaliansubject; the carrier or scaffold material employed; and the purity andpotency of different NELL peptides. In one embodiment, the even morepreferable optimal dosage ranges of NELL peptides includes but are notlimited to 1 ng/mm² to 100 ng/mm², or even more preferably from 100ng/mm² to 1000 ng/mm², or even more preferably from 1 μg/mm² to 100μg/mm², or even more preferably from 100 μg/mm² to 1000 μg/mm². Inanother embodiment, the even more preferable optimal dosage ranges ofNELL peptides includes but are not limited to 1 ng/ml to 100 ng/ml, oreven more preferably from 100 ng/ml to 1000 ng/ml, or even morepreferably from 1 μg/ml to 100 μg/ml, or even more preferably from 100μg/ml to 1000 μg/ml. In yet another embodiment, even more preferableoptimal dosage ranges of NELL peptide for cartilage formation or repairgenerally ranges from 1 μg/kg to 100 μg/kg, or even more preferably from100 μg/kg to 1000 μg/kg, or even more preferably from 1 mg/kg to 100mg/kg with or without a particular carrier or scaffold. Furthermore, itis understood that all dosages can be continuously given or divided intodosages given per a given timeframe. Examples of timeframes include butare not limited to every 1 hour, 2 hour, 4 hour, 6 hour, 8 hour, 12hour, 24 hour, 48 hour, or 72 hour, or every week, 2 weeks, 4 weeks, orevery month, 2 months, 4 months, and so forth. As used herein, the term“significantly above the optimal range” means, e.g., about 1% to about50%, about 5% to about 50%, about 10% to about 50%, about 20% to about50%, about 30% to about 50%, or about 40% to 50% over the optimal range.

The dosage for inhibitors of NELL peptides varies according to the typeof the inhibitor, the bone or cartilage condition to be treated,prevented, or ameliorated, and the age, the location, and the gender ofthe mammalian subject receiving the composition containing theinhibitor. Generally, the dosage for inhibitors of NELL peptides rangesfrom but at not limited to: 0.001 pg/mm² to 1 pg/mm², or more preferablyfrom 0.001 ng/mm² to 1 ng/mm², or more preferably from 0.001 μg/mm² to 1μg/mm², or more preferably from 0.001 mg/mm² to 1 mg/mm², or morepreferably from 0.001 g/mm² to 1 g/mm², with or without a particularcarrier or scaffold. In another embodiment, the dosage for inhibitors ofNELL peptides generally ranges from 0.001 pg/ml to 1 pg/ml, or morepreferably from 0.001 ng/ml to 1 ng/ml, or more preferably from 0.001μg/ml to 1 μg/ml, or more preferably from 0.001 mg/ml to 1 mg/ml, ormore preferably from 0.001 g/ml to 100 g/ml, with or without aparticular carrier or scaffold. In yet another embodiment, the dosagefor inhibitors of NELL peptides generally ranges from 0.001 pg/kg to 1pg/kg, or more preferably from 0.001 ng/kg to 1 ng/kg, or morepreferably from 0.001 μg/kg to 1 μg/kg, or more preferably from 0.001mg/kg to 1 mg/kg, or more preferably from 0.001 gm/kg to 1 gm/kg, morepreferably from 0.001 kg/kg to 1 kg/kg with or without a particularcarrier or scaffold. Furthermore, it is understood that all dosages canbe continuously given or divided into dosages given per a giventimeframe. Examples of timeframes include but are not limited to every 1hour, 2 hour, 4 hour, 6 hour, 8 hour, 12 hour, 24 hour, 48 hour, or 72hour, or every week, 2 weeks, 4 weeks, or every month, 2 months, 4months, and so forth.

The dosage for modulators of receptors of NELL peptides varies accordingto the type of the inhibitor, the type of receptor, the bone orcartilage condition to be treated, prevented, or ameliorated, and theage, the location, and the gender of the mammalian subject receiving thecomposition containing the modulators of receptors of NELL peptides.Generally, the dosage for modulators of receptors of NELL peptidesranges from but at not limited to: 0.001 pg/mm² to 1 pg/mm², or morepreferably from 0.001 ng/mm² to 1 ng/mm², or more preferably from 0.001μg/mm² to 1 μg/mm², or more preferably from 0.001 mg/mm² to 1 mg/mm², ormore preferably from 0.001 g/mm² to 1 g/mm², with or without aparticular carrier or scaffold. In another embodiment, the dosage formodulators of receptors of NELL peptides generally ranges from 0.001pg/ml to 1 pg/ml, or more preferably from 0.001 ng/ml to 1 ng/ml, ormore preferably from 0.001 μg/ml to 1 μg/ml, or more preferably from0.001 mg/ml to 1 mg/ml, or more preferably from 0.001 g/ml to 100 g/ml,with or without a particular carrier or scaffold. In yet anotherembodiment, the dosage for modulators of receptors of NELL peptidesgenerally ranges from 0.001 pg/kg to 1 pg/kg, or more preferably from0.001 ng/kg to 1 ng/kg, or more preferably from 0.001 μg/kg to 1 μg/kg,or more preferably from 0.001 mg/kg to 1 mg/kg, or more preferably from0.001 gm/kg to 1 gm/kg, more preferably from 0.001 kg/kg to 1 kg/kg withor without a particular carrier or scaffold. Furthermore, it isunderstood that all dosages can be continuously given or divided intodosages given per a given timeframe. Examples of timeframes include butare not limited to every 1 hour, 2 hour, 4 hour, 6 hour, 8 hour, 12hour, 24 hour, 48 hour, or 72 hour, or every week, 2 weeks, 4 weeks, orevery month, 2 months, 4 months, and so forth.

Dosage Form

The therapeutically effective dose of an agent included in the dosageform can be selected by considering the type of agent selected and theroute of administration. The dosage form can include a agent incombination with other inert ingredients, including adjutants andpharmaceutically acceptable carriers for the facilitation of dosage tothe patient, as is known to those skilled in the pharmaceutical arts.

In one embodiment, the invention can include a method of treating apatient to induce cartilage formation, comprising administering NELL1peptide at a therapeutically effective dose in an effective dosage format a selected interval to enhance cartilage formation or repair. Themethod of can further comprise administering at least one secondaryagent in the region where cartilage formation or repair is desired,including but not limited to TGF-beta, BMP2, BMP4, BMP7, bFGF, VEGF,PDGF, collagen, bone, bone matrix, tendon matrix or ligament matrix,chondrogenic or osteoblastic cells.

In one embodiment, a method of treating a patient to induce cartilageformation or repair can include harvesting mammalian chondrogenic cells,increasing the concentration of expression of NELL1 peptide in contactwith the chondrogenic cells and administering the chondrogenic cells toa region where cartilage formation or repair is desired.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Injectable Devices

An injectable device containing NELL (with or without cells) can bedirectly injected into spinal discs to promote cartilage formation. Adisc nucleus replacement device impregnated with NELL is designed toreplace the inner portion of the vertebral disc (the nucleus) or boththe inner and outer portion of the disc. An injectable device containingNELL (with or without cells) can be directly injected into the variousjoint spaces (e.g., knee, temporomandibular joint, wrist) or implantedarthroscopically or openly into various joint spaces.

Example 2 Cartilage Differentiation, Maturation and Hypertrophy WithoutNecessarily Mineralization

NELL1 transgenic overexpression mice were created with the rationale wasthat NELL1 overexpression transgenic mice would exhibit alteredintramembranous or endochondral bone formation. The invention was testedwith F2 progeny from NELL1 transgenic mice. Histology from various formsof NELL1 overexpression mice has demonstrated increased cartilagedifferentiation, maturation, and hypertrophy without necessarilymineralization in both hyaline cartilage areas (FIG. 1) andfibrocartilage areas (FIGS. 2A-2F).

Goat auricular cartilage was minced to 1×3 mm pieces and digested with0.25% trypsin/1 mM EDTA at room temperature for 30 min, followed by 3mg/ml collagenase II (Sigma, St Louis, Mo., USA) digestion with shakingat 37 C for 6 h. The cell suspension was filtered through a 70 mmstrainer and the chondrocytes were then pelleted by centrifugation.After washing with PBS, the cells were cultured in DMEM (Gibco BRL,Grand Island, N.Y., USA) plus 10% fetal calf serum (Hyclone, Logan,Utah, USA), 100 U/ml penicillin and 100 mg/l streptomycin at 37° C. with5% CO2. The cells were then treated/transduced with AdNELL1, AdBMP2, orAdLacZ. The in vitro transduction efficiency was assessed by stainingfor beta galactosidase (FIG. 3). The cells were combined with pluronicF127 (Sigma) as a common carrier for nude mice subcutaneous injection/orimplantation and then examined at 2 weeks (FIG. 6) or 4 weeks (FIGS.4,5,7-9). A total of 8 million cells were injected/implanted per site.

High-resolution micro-computed tomography (microCT), which utilized 9-20μm resolution technology from μCT40 (Scanco Medical, Basserdorf,Switzerland) was performed on 4 week samples (FIG. 5). MicroCT data werecollected at 55 kVp and 145 μA and reconstructed using the cone-beamalgorithm supplied with the microCT scanner by Scanco. Visualization andreconstruction of the data were performed using the μCT Ray T3.3 and μCTEvaluation Program V5.0 provided by Scanco Medical.

Harvested samples were processed and embedded in paraffin wax. Sixmicron-thick sections, using a microtome (McBain Instruments,Chatsworth, Calif.), were placed on poly-L-lysine-coated Polysinemicroscope slides (Erie Scientific Company, Portsmouth, N.H.) and bakedat 37° C. overnight. Samples were hematoxylin and eosin (H&E) stained.Additional analysis utilized alcian blue staining. Sections were stainedwith alcian blue solution for 30 min followed by washing in 3% glacialacetic acid followed by water. Sections were then counterstained withnuclear fast red solution and rinsed in distilled water. Finally,sections were dehydrated in alcohol and cleared in xylenes beforemounting in permount (FIGS. 6 and 7).

Six-micron-thick sections were dewaxed in xylenes and rehydrated inethanol baths. Sections were enzyme-treated for antigen retrieval with20 μg/ml Proteinase K at 37° C. for 10 min and then blocked with 5%horse serum for 2 h at room temperature. Sections were incubated withappropriate primary antibodies at 4° C. overnight then incubated with abiotinylated anti-rabbit IgG secondary antibody (Vector Laboratories,Burlingame, Calif.) for 1 h at room temperature. Positiveimmunoreactivity was detected using Vectastain ABC reagents and AECchromagen (both from Vector Laboratories) according to themanufacturer's instructions. Controls for each antibody consisted ofincubation with secondary antibody in the absence of primary antibody.Sections were counterstained with hematoxylin for 2 min followed by 10min in running water. Aqueous mounting medium was used with cover slips.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art thatchanges and modifications can be made without departing from thisinvention in its broader aspects. Therefore, the appended claims are toencompass within their scope all such changes and modifications as fallwithin the true spirit and scope of this invention.

1. A method of increasing cartilage formation or repair comprising:applying a NELL-1 peptide or an expression vector encoding said NELL-1peptide in an effective amount for cartilage formation or repair in aregion where cartilage formation or repair is desired; optionallyapplying an agent to the region where cartilage formation or repair isdesired; and at least inducing hypertrophy of chondroblast in the regionwhere cartilage formation or repair is desired, wherein the NELL-1peptide comprises the amino acid sequence set forth in SEQ ID NO: 2, 4,or 6 or a fragment of the amino acid sequence of SEQ ID NO: 2, 4, or 6which has cartilage-forming activity.
 2. The method of claim 1, whereincartilage formation or repair includes bone healing or boneregeneration.
 3. The method of claim 1, wherein the agent is selectedfrom chondroprotective agents, anti-pain and/or anti-inflammatoryagents, growth factors, cytokines, small molecules, anti-angiogenicfactors, and combinations thereof.
 4. The method of claim 1, wherein theagent is selected from collagen, bone matrix, ligament matrix, tendonmatrix, chondrogenic cells, and osteochondroprogenitor cells.
 5. Amethod of treating or ameliorating a cartilage related condition,comprising applying to a site in a mammalian subject a compositioncomprising a NELL-1 peptide or an expression vector encoding said NELL-1peptide in an effective amount for cartilage formation or repair;wherein the NELL-1 peptide comprises the amino acid sequence set forthin SEQ ID NO: 2, 4, or 6 or a fragment of the amino acid sequence of SEQID NO: 2, 4, or 6 which has cartilage-forming activity.
 6. The method ofclaim 5, wherein the composition further comprises a second agentselected from the group consisting of chondroprotective agents,anti-pain and/or anti-inflammatory agents, growth factors, cytokines,small molecules, anti-angiogenic factors and combinations thereof.
 7. Amethod of treating or ameliorating a cartilage related condition,comprising applying to a site in a mammalian subject an implantcomprising a substrate having a surface, wherein at least a portion ofthe surface includes a composition that comprises a NELL-1 peptide or anexpression vector encoding said NELL-1 peptide in an effective amountfor cartilage formation or repair; wherein the NELL-1 peptide comprisesthe amino acid sequence set forth in SEQ ID NO: 2, 4, or 6 or a fragmentof the amino acid sequence of SEQ ID NO: 2, 4, or 6 which hascartilage-forming activity.
 8. The method of claim 7, wherein thecomposition further comprises a second agent selected from the groupconsisting of chondroprotective agents, anti-pain and/oranti-inflammatory agents, growth factors, cytokines, small molecules,anti-angiogenic factors and combinations thereof.